Metastatic melanoma remains a highly lethal malignancy, and despite advances in targeted treatments, new therapeutic options are required. The overarching goal of this project is to develop a cell-based therapeutic to deliver immunostimulatory cytokines directly into tumors, which is a promising therapeutic strategy that cannot yet be achieved with existing drugs and delivery vehicles. Monocytes and macrophages, key components of the body's innate immune system, readily infiltrate tumors and are safe in humans. This makes them ideal vehicles for the local delivery of immunotherapies that carry significant toxicities when infused systemically. Whereas constant cytokine production from engineered cells could also be toxic, limiting secretion of such cytokines to the tumor site could best balance safety and efficacy. To this end, the Leonard Lab has developed the Modular Extracellular Sensory Architecture (MESA), a synthetic receptor system that can be used to engineer cells to respond in defined ways to specific environmental cues. Notably, MESA can be used to engineer cells to recognize Vascular Endothelial Growth Factor (VEGF), a marker of the tumor environment in many cancers, and in response, produce interleukin (IL)-2, a cytokine currently used to treat several cancers. This conditional VEGF-activated, MESA-induced expression of cytokines from monocytes and macrophages would increase the specificity of cytokine delivery for the tumor environment. We therefore propose delivering anti-tumor cytokines into tumors in large local doses, beyond those that can be safely achieved by systemic administration, by using MESA-engineered mononuclear cells (EMNs). This project aims to confer a novel behavior on the innate immune system - the ability to recognize tumor markers and in response produce pro-inflammatory cytokines, driving the body's own immune system to attack the tumor. The first proposed aim will evaluate the ability of the MESA EMNs to detect VEGF in vitro and in vivo, utilizing a MESA that induces transcription of a bioluminescent reporter when the EMNs recognize VEGF. The second aim focuses on developing the intracellular machinery required to engineer MESA EMNs that produce IL-12 and synergistic cytokines in response to VEGF. In the third aim, we will infuse these cytokine secreting MESA EMNs into mice with melanoma, in which tumors contain common human melanoma mutations, and assess the effects of the EMNs on tumor growth and mouse survival in order to evaluate the therapeutic potential of the EMNs. If successful, this technology will be able to provide an a new therapeutic approach, or provide an adjuvant therapeutic to boost the efficacy of current treatment protocols with cytokines, checkpoint inhibitors, or other cellular therapy strategies, for melanoma, a disease which remains incredibly deadly and still in need of new therapeutic options.